(19)
Europdisch^Mtentamt
European Patent Office
Office europ^en des brevets
(11)
EP 0 633 030 B1
(12)
EUROPEAN PATENT SPECIFICATION
(45) Date of publication and mention
of the grant of the patent:
28.04.1999 Bulietin1999/17
(21) Application number: 94304902.3
(22) Date of filing: 04.07.1994
(51) IntCL^: A61K 49/00
(54) The preparation of protein encapsuiated insoluble gas microspheres.
Die Herstellung von wasserunlOslichen Proteinverl^apseiten gasenthaltenden Mil^osphdren,
La preparation de microspheres k gaz encapsul^es par une proteine insoluble.
m
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CO
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CO
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CD
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Q.
UJ
(84) Designated Contracting States:
AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL
PTSE
(30) Priority: 02.07.1993 US 86717
26.01.1994 US 187656
(43) Date of publication of application:
11.01.1995 Bulletin 1995/02
(60) Divisional application:
98116817.2 / 0 885 615
(73) Proprietor:
MOLECULAR BIOSYSTEMS, INC.
San Diego California 92121 (US)
(72) Inventors:
• Lambert, Karel J.
San Diego, California 92126 (US)
• Podeil, Sheila Benay
San Diego, California 92122 (US)
• Jabionsld, Edward G.
Escondido, California 92029 (US)
• Hulle, Carl
San Diego, California 92109 (US)
• Hamilton, Kenneth
Cardiff, California 9200 (US)
• Lohrmann, Rolf
La Jolla, California 92037 (US)
(74) Representative:
Goldin, Douglas Michael et al
J.A. KEMP & CO.
14 South Square
Gray's Inn
London WC1R5LX(GB)
(56) References cited:
EP-A- 0 224 934
EP-A- 0 359 246
WO-A-92/05806
EP-A- 0 324 938
EP-A- 0 554 213
WO-A-93/05819
Remarks:
The file contains technical information submitted
after the application was filed and not included in
this specification
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give
notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in
a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art.
99(1) European Patent Convention).
EP0 633 030B1
Description
Technical Field
5 [0001] This invention relates to the preparation of ultrasonic imaging agents composed of proteinaceous micro-
spheres encapsulating insoluble gases and methods for their production and use.
10 [0002] Diagnostic ultrasonic imaging is based on the principle that waves of sound energy can be focused upon an
area of interest and reflected in such a way as to produce an image thereof. An ultrasonic scanner is placed on a body
surface overlying the area to be imaged, and ultrasonic energy in the form of sound waves are directed toward that
area. The scanner detects reflected sound waves and translates the data into video images. When ultrasonic energy is
transmitted through a substance, the amount of energy reflected depends upon the velocity of the transmission and the
15 acoustic properties of the substance. Changes in the substance's acoustic properties (e.g., variations in acoustic
impedance) are most prominent at the interfaces of different acoustic densities, such as liquid-solid or liquid-gas. Con-
sequently, when ultrasonic energy is directed through tissue, organ structures generate sound reflection signals for
detection by the ultrasonic scanner. These signals can be intensified by the proper use of a contrast agent.
[0003] Ultrasound imaging agents of particular importance employ the use of gas because of its efficiency as a reflec-
20 tor of ultrasound. Resonant gas bubbles scatter sound a thousand times more efficiently than a solid particle of the
same size. Ophir and Parker describe two types of gas-containing imaging agents as being: (1) free air bubbles and (2)
encapsulated air bubbles (Ultrasound in Medicine and Biology 15 (4):319-333. 1989). However, free gas bubbles of the
appropriate size are too short-lived to be effective for most in vivo applications (Meltzer, et al.. Ultrasound in Medicine
and Biology S:263-269, 1980). Ophir and Parker point out that the development of encapsulated gas bubbles was an
25 attempt to overcome this problem.
[0004] The second major class of gas-containing ultrasound contrast agents described by Ophir and Parker are the
encapsulated microbubbles, hereinafter referred to as "microspheres". The gas bubble is surrounded by a shell com-
posed of a protein or other biocompatible material. A current commercial microsphere contrast agent is ALBUNEX®
(Molecular Biosystems, Inc., San Diego, CA) which is composed of human serum albumin encapsulated air micro-
30 spheres. See U.S. Patent Nos. 4,572,203 and 4,844,882.
[0005] Air microspheres have been shown to quickly lose echogenicity when subjected to pressures of 150 mm Hg.
such as would be encountered during injection and circulation in vivo (deJong, N. et al.. Ultrasound Med. Biol. 19:279-
288, 1993). Present encapsulating technology has yet to produce a material suitable as an ultrasound contrast agent
that will survive long enough in vivo for most desired applications. In fact, an agent capable of imaging the myocardial
35 wall must withstand transient pressures pulses of at least 250 mm Hg (about 5 psig).
[0006] In an effort to solve the pressure-instability problem of microspheres, recent teachings have centered on
improving the shell, because, it is believed, the microsphere shells or "membranes" are too fragile or brittle under pres-
sure, resulting in rapid collapse in vivo. Giddey (PCT/EP91/01 706; PCT 92/05806) stated, "because of their rigidity, the
membranes cannot sustain sudden pressure variations to which the microspheres can be subjected, for instance during
40 travel through the bloodstream, these variations or pressure being due to heart pulsations." To overcome shell rigidity,
he proposed to pre-emulsify air in a protein solution containing a large percentage of a viscosifying agent (40%-80%
polyols) and subject it to mechanical shear in a high speed blender. Bubbles of the appropriate size are collected and
coated with a suitable surfactant to stabilize them in a soft shell.
[0007] Holmes (PCT WO 92/1 721 3) proposed to enhance the in vivo stability of protein microspheres by strengthen-
45 ing the shell with biodegradable chemical crosslinking reagents.
[0008] Bichon et al. (EPA 90/810367) and Schneider et al. ( Inv. Radiol. 27:134-139. 1992) describe the production of
porous (5 to 2000 nm pore size) polymeric "microballoons". They report in the European Patent /Application that "the
microporous structure of the microballoon's envelope is a factor of resiliency, i.e., the microspheres can readily accept
pressure variation without breaking."
50 [0009] Erbel and Zotz (U.S. Patent No. 5,190,982) describe a cross-linked polymeric microcapsule in which air is
entrapped.
[001 0] Schneider et al. (EPA 554,21 3) indicates that the pressure resistance of microspheres can be improved by hav-
ing at least a portion of the gas that is encapsulated be a gas which has a Sgas/MWgas ^ 0.0031 . where Sgas is the water
solubility of the gas in liters/liter and MWggg is the average molecular weight of the gas in daltons. Table 1 of the refer-
55 ence lists N2, SFe, CBrFs and CF4 as meeting this criterion. The reference teaches that these microspheres may be
made in either of two ways. The first is a two-step method in which air-containing microspheres are prepared by a
known method and the air is replaced with the insoluble gas by a gas-exchange method, e.g., by incubating the air-filled
microspheres under an atmosphere of the insoluble gas for an adequate time.
Baqkgrpund
EP0 633 030B1
[001 1 ] The second is a one-step method in which microspheres are made by the method of EPA 324,938 (See Exam-
ple 1 of the reference) using an insoluble gas instead of air. In that method, the gas was passed over a solution of a
shell-forming material (e.g., a solution of albumin) while a sonicator horn was lowered into the vessel and then removed.
[0012] Unfortunately, neither of these methods is practically useful for making stable suspensions of protein encap-
sulated insoluble gas-filled microspheres. Using the first (two-step) method, only a small number of air-filled albumin
microspheres can survive the exposure to an Insoluble gas environment (the second step of the two-step method). The
efflux of soluble gases (air) out of the microspheres is greater than the influx of insoluble gas into the microspheres
which results in complete collapse of the microspheres, leaving only shell debris. This effect is particularly pronounced
with the more insoluble gases such as pert luoroethane. The second process produces microspheres which lose volume
as pressure is applied and do not exhibit recovery after the release of pressure. It is believed that both of these proc-
esses produce inferior microspheres because the microspheres contain significant amounts of air and that the pres-
ence of air during formation may lessen the benefits of producing the microspheres in the presence of insoluble gas
alone. It is noted, in this regard, that prior investigators have considered the presence of oxygen essential in making
microspheres by cavitation.
[001 3] Suslick reported that ultrasound-associated cavitation was suitable as a method of production of microspheres
only in the presence of oxygen. In detailed studies, Suslick et al., (Proc. Natl. Acad. Sci. 88:7708-7710, 1991; J. Am.
Chem. Soc. 112:7807-7809, 1990) reported that oxygen participates in the cavitation-induced intermolecular rear-
rangement of disulfide bonds needed for a stable protein shell. Suslick states "We find that microcapsule formation is
strongly inhibited by the absence of O2". He goes on to state, "If the reaction is run under an inert atmosphere (He, Ar
or N2), microcapsules are not formed." "Experimentally, high concentrations of microbubbles are synthesized only when
the reaction is run under O2 or air." See also, U.S. 4.774,958. The previous belief that air was necessary for the forma-
tion of albumin microspheres was also noted in Holmes (PCT WO 92/17213). Therein, the production of microspheres
containing various low molecular weight gases was disclosed. However, when describing the production of albumin
microspheres by sonication, the authors stated. "Another well-established method described, i.e., US-A-4,774,958, for
creating a gas-containing bubble is by sonication of the mixture in the presence of air. " (Emphasis added.)
[001 4] In one aspect, the current invention relates to the unexpected finding that high concentrations of proteinaceous
microspheres, entrapping relatively insoluble gas, can be made by ultrasound or mechanical cavitational processes in
the presence of the insoluble gas without the presence of oxygen. Such microspheres exhibit surprising and greatly
improved stability and elasticity to applied pressure, with better or equivalent echogenicity. The proteinaceous shell pre-
vents coalescence and resists expansion due to diffusion of dissolved atmospheric gases from the surrounding envi-
ronment.
[001 5] In another aspect, the current invention relates to a novel procedure for the production of protein-shelled micro-
spheres which employs mechanical energy in the form of shear forces. These forces are responsible for mechanically
shearing a liquid-gas mixture to form a microbubble suspension, and also for causing hydrodynamic cavitation which
releases energy This energy can be absorbed by the surrounding liquid to effect localized protein denaturation and
deposition at a gas-liquid interface to form discrete microspheres. Hydrodynamic cavitation can be distinguished from
ultrasonic (acoustic) cavitation on the basis of the way in which each produces pressure variations on liquid systems
which lead to the release of energy In the former, pressure variations are produced by the rapid flow of a liquid through
an orifice or across a surface, whereas in the latter, cycles of high frequency sound waves produce rapid local pressure
variations. (See R Ron Young. 1989 Cavitation Pages 4-5, McGraw-Hill Book Co. London). Additionally, hydrodynamic
cavitation is produced in a flowing liquid, i.e., a liquid which is flowing through or across a stationary object. In compar-
ison, acoustic cavitation is produced in a liquid system which must remain stationary through enough cycles of increas-
ing and decreasing pressure (positive and suction pressure) to exhibit cavitation. Even in a continuous flow sonication
system such as that described in U.S. Patent No. 4,957,656, the residence time in an acoustic cavitation process makes
it harder to control than in a true single-pass hydrodynamic cavitation system such as is described by the present inven-
tion.
[001 6] Microbubble suspensions produced by mechanical shear forces are used per se as contrast agents or formed
into microspheres by further processing. For instance, PCT Publication No. WO 92/05806 describes the preparation of
a microbubble suspension, which they refer to as a "loam", of a f ilmogenic protein which is prepared by whipping a pro-
tein solution containing viscosifiers into a coarse foam at a constant temperature below that which would denature the
protein. The resultant foam is then sheared mechanically to form bubbles of a desired range which are stabilized by the
presence of the viscosifiers. The bubbles may then be further processed into microspheres by heat denaturation or by
the addition of cross-linkers to harden the protein film surrounding the bubbles.
[0017] European Patent Application Pub. No. 0 450 745 Al describes a process for making microspheres by forming
an oil-in-water emulsion by mechanical shearing and simultaneously or subsequently adding a water-insoluble polymer
which deposits at the interface. The hydrophobic phase is then evaporated to form air or gas filled microspheres.
[0018] Thus, the present invention also relates to an improved method for making microspheres from heat-denatura-
ble protein by subjecting a protein solution to mechanical shear forces. Such forces create a suspension of microbub-
EP0 633 030B1
bles that are simultaneously or subsequently encapsulated by a discrete shell. Becausebf the nature of cavitational
heating, the denaturation of the protein is localized and forms the shell by depositing at the liquid-gas interface. This
new method is easier to scale-up and leads to improved product yields as conrpared to the prior acoustic-based meth-
ods of making microspheres.
Disclosure of the Invention
[0019] One aspect of the invention is a method of making encapsulated gas microspheres useful as an ultrasonic
imaging agent, the method conprising subjecting a mixture of an aqueous solution of a f itmogenic protein and a phar-
10 macologically acceptable water insoluble gas to ultrasonic or mechanical cavitation in the absence of oxygen, the gas
having a solubility of less than: 0.01 mL gas per mL of water (at 25°C).
[0020] These encapsulated gas microspheres are useful as an ultrasonic imaging agent.
Brief PescrjptiQn Of the Prawings
15
[0021]
Fig. 1 is an exploded schematic view of one type of mill (a Gaulin mill) that may be used in the mechanical cavitation
process of the invention;
20 Fig. 2 is an exploded schematic view of another type of mill (a Bematek mill) that may be used in the mechanical
cavitation process of the invention ;
Fig. 3 is an exploded schematic view of still another type of mill (a Silverson mill) that may be used in the mechan-
ical cavitation process of the invention.
Fig. 4a shows the pressure resistance of air-filled albumin microspheres. A microsphere suspension was placed in
25 a syringe and pressurized at 40 psig 2.76 bar. Particle distributions before and after pressurization are shown.
Fig. 4b shows the pressure resistance of perf luoropropane*f illed albumin microspheres. A microsphere suspension
was placed in a syringe and pressurized at 40 psig 2.76 bar. Particle distributions before and after pressurization
are shown.
Fig. 4c shows the pressure resistance of perf luoroethane-f illed albumin microspheres. A microsphere suspension
30 was placed in a syringe and pressurized at 40 psig (2.76 bar). Particle distributions before and after pressurization
are shown.
Fig. 4d shows the pressure resistance of sulfur hexaf luoride-filled albumin microspheres. A microsphere suspen-
sion was placed in a syringe and pressurized at 40 psig (2.76 bar). Particle distributions before and after pressuri-
zation are shown.
35 Fig. 4e shows the pressure resistance of argon-filled albumin microspheres. A microsphere suspension was placed
in a syringe and pressurized at 40 psig (2.76 bar). Particle distributions before and after pressurization are shown.
Fig. 5 shows the pressure resistance of dilute suspensions of microspheres at 3.0 psig (0.21 bar). A diluted sus-
pension of microspheres were placed in a 1 cm cuvette and subjected to 3.0 psig (0.21 bar) at the time t=30 sec-
onds. Shown are data for perfluoroethane, perfluoropropane, sulfur hexaf luoride and air microspheres.
40 Fig. 6 shows pressure resistance of dilute suspension of argon microspheres at 3.0 psig (0.21 bar). Diluted argon
microspheres were placed in a 1 cm cuvette and subjected to 3.0 psig (0.21 bar) at time t=30 seconds.
Fig. 7 shows the effect of degassed buffer on microspheres. Microspheres were added to increasing amounts of
degassed buffer, with mixing, and the mixture brought to constant volume for determination of concentration. Data
for air. perfluoropropane. perfluoroethane. and sulfur hexafluoride microspheres are plotted versus volume of
45 degassed buffer.
Fig. 8 shows graphs of data described in Example 1 1 , infra.
Modes for Carrying Out the Invention
50 [0022] The microspheres are formed in an aqueous suspension from the ultrasonic or mechanical cavitation of aque-
ous solutions of filmogenic proteins in the presence of an insoluble gas and in the substantial absence of oxygen, i.e..
under anaerobic (closed system) conditions. The microspheres are echo reflective and of a size suitable for transpul-
monary passage, with a mean diameter less than 10 microns and greater than 0.1 microns. The size distribution may
be altered by fractionation into larger or smaller microsphere populations. The microspheres may or may not be con-
55 centrated by removal of excess aqueous phase, or collected and resuspended in a second aqueous solution.
[0023] The gas used to make these novel microspheres need only be pharmacologically acceptable and insoluble in
the aqueous media in which they are placed (i.e., initially the medium in which they are made and, when used, in the
blood). Solubility in water is a close approximation of solubility in such media. The term "gas" refers to any compound
EP0 633 030B1
which is a gas or capable of forming gas at the temperature at which imaging is being performed (typically normal phys-
iological temperature). The gas may be composed of a single compound or a mixture of compounds. Appropriate gases
would include, but are not limited to, fluorine-containing gases such as sulfur hexaf luoride, perfluoroethane, perf luoro-
propane. perf tuoromethane, and perfluorobutane. Solubility of a gas can be defined by determining the Bunsen Coeffi-
5 cient of the gas of interest. TTiis value is the volume of gas which is absorbed by a unit volume of solvent, (see Wen, W-
Y, Muccitelli. JA. J. Sol. Chem. 8:225-240 (1979)). Gas suitable for use in the present invention should have a Bunsen
Coefficient in water at 25''C of less than 0.01 mUmL of solution. Table 1 gives the Bunsen Coefficients of several gases
BUNSEN COEFFICIENTS OF GASES IN
WATER (1 atmosphere, mL/mL)
Gas
25*0
Carbon Dioxide
1.383
0.824
Argon
0.047
0.033
Oxygen
0.043
0.031
Nitrogen
0.021
0.016
Sulfur hexaf luoride
0.008
0.0054
Perfluoromethane
0.0082
0.00504
Perfluoroethane
0.0027
0.00138
Perfluoropropane
0.0016
N/A
Perfluorobutane
0.0007
N/A
[0024] Another characteristic of the gas contained in the microspheres is that the diffusivity of the gas is less than 4
30 X 1 0"^ cm^/sec at 25**C in water. It should be noted, however, that the diffusivity constant varies in different solvents and
at different temperatures, but for purposes of selecting a gas, the gas should meet this criterion.
[0025] Pharmacologically acceptable refers to the property that the selected gas must be biocompatible and have
minimal toxicity.
[0026] Perfluoropropane is preferred because it provides an insoluble gas that (1) will not condense at the tempera-
35 tures of manufacture and use. (2) has no isomeric forms, (3) produces microspheres that exhibit excellent pressure
resistance, and (4) is pharmacologically acceptable.
[0027] The gas miaobubble is encapsulated by a f ilmogenic protein shell. The term f ilmogenic refers to the ability of
the protein to form a shell around the entrapped gas. with hydrophilic groups oriented externally and hydrophobic
groups oriented internally, upon protein insolubilization (caused by heat denaturation). The protein will necessarily have
40 both hydrophilic and hydrophobic amino acids. Suitable proteins will include naturally occurring proteins such albumin,
gamma-globulin (human), apo-transferrin (human), b-lactoglobulin. and urease. Although naturally occurring proteins
are preferred synthetic proteins (homopolymeric or heteropolymeric) which exhibit tertiary structure and are susceptible
to heat denaturation may be used. Particularly well suited tor the present invention is albumin, and more particularly,
human albumin. The protein is present in the solution at a concentration in the range of about 0. 1 to 1 0% w/v, preferably
45 about 1 to 5% w/v, and most preferably about 1% w/v.
[0028] Proteins suitable for the present invention, or the resulting microspheres, may be chemically modified for the
purpose of organ targeting or quenching immunogenic activity (e.g., modification with polyethylene glycol). However,
the present invention does not involve addition of chemical crosslinking agents, or additional modifications to the pro-
teins for the purpose of forming the microspheres.
50 [0029] The microspheres of the present invention are formed by insolubilizing portions of a protein in solution as a
result of cavitation in the presence of an insoluble gas, without the presence of oxygen (i.e.. in a closed system in which
air contamination is avoided.) Such protein insolubilization is characterized primarily by local protein denaturation and
orientation around the gas core, the latter of which may be enhanced in the presence of the insoluble gas.
[0030] The system used to heat-insolubilize the protein for formation of the microspheres of the present invention
55 must be anaerobic, i.e., closed to the atmosphere, and is referred to as a "closed system". In comparison, an "open sys-
tem" is one which is open to the atmosphere. The gas entrapped in microspheres made in such a closed system will
necessarily contain only the insoluble gas used for formation. Contamination by atmospheric gases can be monitored
using an O2 electrode to measure the presence of O2 in the system effluent. In the present invention, microspheres are
EP0 633 030B1
produced which initially contain only the gas used for formation. However, when the gas content is determined experi-
mentally, there is a certain amount of unavoidable contamination by the atmospheric gases during the experimental
procedure, thus causing the amount of resultant gas measured to be less than 100%. Accordingly, measurements of
gas greater than 85% are representative of microspheres whose initial gas content was entirely insoluble gas.
5 [0031] After formation of the microspheres, exposure to the atmosphere should be avoided during packaging. For
example, microspheres should be sealed in vials or other air-tight containers within 5 to 30 seconds from the time they
exit from the closed system. Additionally, any head space in the vials should be removed and replaced with the gas
used in formation during packaging.
[0032] Filled with an insoluble gas, these protein microspheres exhibit remarkable stability, surviving exposure of 40
10 psig {>2000 mm Hg, 2.76 bar) at a concentration of about 1.0 x 10^ microspheres per mL The microspheres also
exhibit elasticity in a dilute suspension, exhibiting compression under pressure of 3-10 psig (0.21 to 0.69 bar) and
returning to the original volume upon the release of pressure. Additional chemical cross-linking would be disadvanta-
geous because the resultant microspheres would have too rigid a structure to exhibit enhanced pressure stability.
[0033] The microspheres of the present invention, unlike free microbubbles, are resistant to coalescence and diffusion
15 driven expansion. Microspheres containing insoluble gas incubated in air or oxygen saturated solution, at various tem-
peratures, do not increase in mean diameter or increase in total volume. The protein shell, although elastic when sub-
jected to pressure, is strong enough to resist expansion or tearing by gas diffusion or exchange. The presence of the
protein shell prevents coalescence, and maintains the gas in small, individual bubbles for up to several months, similar
to air-filled protein microspheres. The inability of insoluble gas-filled microspheres to measurably expand by exchange
20 with solvated atmospheric gases is a novel and key property for the use of this material as an ultrasound agent.
[0034] Insoluble gas-entrapped protein microspheres exhibit resistance to collapse upon exposure to degassed aque-
ous solutions. Unlike free microbubbles or encapsulated microspheres filled with air, insoluble gas-filled microspheres
may be added to vacuum degassed water and maintain integrity at high dilution. Air-filled material collapses in blood
due to the efflux of the oxygen component of the gas phase. The ability of microspheres filled with an insoluble gas to
25 resist collapse in a partially degassed or pressurized environment increases dramatically the duration of ultrasound
contrast in vivo.
[0035] The microspheres of the present invention are produced by ultrasound or mechanical cavitation. A process of
ultrasound production of air-filled microspheres has been described by Cerny (USP 4,957.656).
[0036] Mechanical cavitation is one process for making the novel insoluble gas-filled microspheres of this invention.
30 It may also be used to make air-filled or soluble gas (e.g., N2, H2, argon)-filled microspheres.
[0037] In the novel mechanical cavitation procedure of the present invention, the aqueous solution of heat-denatura-
ble protein is provided at a temperature necessary to achieve incipient denaturation temperature during the subsequent
mechanical emulsrfication of the solution. The denaturation temperature of the protein in solution will normally be in the
range of 50 to 100°C. It can be obtained from tables of thermal protein denaturation in the literature, or experimentally
35 by any known method. For example, to determine the denaturation temperature experimentally, a protein solution can
be heated in a water bath while stirring. The denaturation temperature is the temperature at which insoluble material is
first observed. Note that the denaturation temperature is affected by the nature, purity and source of the protein, the
concentration of protein in the solution, the pH, buffer, ionic strength, the presence of stabilizers and the presence of
chemical denaturants or detergents. Therefore, it is necessary to determine the denaturation temperature of the protein
40 in the environment in which it will be employed to make microspheres. If desired, additives such as detergents or polar
solvents can be employed to change the temperature at which denaturation takes place.
[0038] Table 2 gives the denaturation temperatures of several naturally occurring proteins which were determined
experimentally as described above:
45
50
55
EP0 633 030B1
Table 2
PROTEIN
CONCENTRATION
pH
SOLVENT
''^denaturation
MiimAn 5^priim AlHiimin
USP Swiss Red Cross
(Bern, Switzerland)
1 1 1^/ 111^
I^Ctwl, *rl 1 IIVI wUUIUill
Caprylate, 4mM Tryp-
tophanate
Human Serum Albumin,
USP Swiss Red Cross
(Bern. Switzerland
10 mg/mL
6.9
0.9% NaCI, 1 mM Sodium
Caprylate, 1 mM Tryp-
tophanate
ys'C
p-Lactoglobulin. Sigma (St.
Louis, MO)
25 mg/mL
7.6
USP Water
sec
ap-Globin, Sigma (St.
Louis. MO)
25 mg/mL
5.0
USP Water
SO'C
Lvsozvme Siama fSt
Louis, MO)
100 mo/mL
1 WW 1 1 'Sv III mam
75
5 mM IRIS* 2mM DTT***
31**C as determined
V 1 w OO Udwl 1 1 III ICU
immediately after addition
of DTT
Muman ^amma r^lohulin
acid pH method. Sigma
(St. Louis, MO)
dOmn/ml
*T w 1 1 IM/ 111^
5 0
10 mM MF5^** nH S 0
i\j 1 1 IIVI ivi^w . H v>v
Human Gamma Globulin,
alkaline pH method, Sigma
(St. Louis, MO)
40 mg/mL
9.8
10 mM TRIS, pH 9.8
eg-c
apo-Transferrin, Sigma (St.
Louis, MO)
20 mg/mL
7.5
10 mM TRIS*
7rc
* TRIS = 2-amino-2-(hydroxymethyl)-1 ,3-propanediol
** MES = 2-(N-morpholino)ethanesulfonic acid
DTT = dithiothreilol
[0039] Each apparatus employed to shear the protein solution/gas mixture will cause a certain amount of additional
heating of the protein solution due to the mechanical shear forces exerted on the solution. That heat must be sufficient
to cause localized denaturation of the protein at the gas-liquid interface. It is thus important to determine the amount of
temperature increase caused by the apparatus so that the temperature at which the protein solution is introduced into
the apparatus can be adjusted to achies/e such local thermal denaturation. Specifically, the bulk temperature of the liq-
uid in the apparatus must coincide with the incipient denaturation temperature immediately prior to cavitation. The cav-
itation event generates the additional heat necessary to locally denature the protein. Incipient denaturation temperature
is defined as the temperature at which the protein is on the verge of denaturation, but the solution does not contain any
denatured protein. This temperature is just below, typically 1 to 5°C below, the denaturation temperature. If necessary,
the starting protein solution may be preheated prior to being introduced into the apparatus to a tennperature that allows
the incipient denaturation temperature to be reached.
[0040] Once the proper starting temperature of the protein solution has been achieved, the solution is combined with
a suitable gas. for example by introducing the gas into the protein solution prior to or during the emulsif ication step at a
volume to volume ratio in the range of about 5% to 200% gas:liquid, preferably about 20% to 100%. The proper gas:liq-
uid ratio will depend on the geometry of the apparatus, the physical characteristics of the gas (solubility, density, molec-
ular weight, etc.). and can be adjusted to optimize output.
[0041] After the gas and protein solution are combined, the mixture is emulsified and subjected to cavitation under
conditions that produce microspheres. This is accomplished using an apparatus in which mechanical shearing and
hydrodynamic cavitation can be produced, such as high speed mixers, mills, f luidizers and the like. A preferred appara-
tus is a colloid mill which is defined as "a machine consisting of a high-speed rotor and a stator, dispersion or emulsifi-
cation being effected by the opposing faces." Advanced Filtration and Separation Technology, p. 108-1 10. Examples of
specific milling apparatuses which can be used are as follows:
Model #2 1/2
- Bematek. Beverly, MA
EP0 633 030B1
Model W250V - Greerco. Hudson. NH
Model 2F - APV Gaulin, Everett, MA
Model L4R - Silverson, Chesham. UK
Model Polytron PT3000 - Kinematioa, Littaw, Switzerland
[0042] When used to make insoluble gas-f ilted microspheres, the colloid mills should be closed to the atmosphere so
as to avoid introducing air into the mixture.
[0043] Figures 1 -3 provide further details of several types of mills that may be used in the mechanical cavitation proc-
ess.
[0044] Fig. 1 depicts the essential elements of a Gaulin mill. These are: a rotating shaft (10) operably connected to a
motor (not shown); a disc rotor (12) affixed to the end of the shaft (10); and a stator (11). Stator (1 1) has a central bore
opening (18) and a counterbore (16) into which the rotor is received. In this mill the protein solution and gas are fed
through the mill via a lee" (15). The protein solution/gas mixture is emulsified and cavitates between the surfaces of
the rotor and stator. The "gap" in this mill is the space between radial surface (1 7) of the stator counterbore and the cir-
cumferential radial surface of the rotor. The temperature of the microsphere product will be taken (e.g. with a thermo-
couple, not shown) as the mixture exits past the stator (11).
[0045] Fig. 2 shows the essential elements of a Bematek mill. This mill is similar in structure and function to the Gaulin
mill of Fig. 1 -- the main difference being the configurations of the rotor and the stator counterbore. It includes a rotary
shaft (20) that carries a frustroconical rotor (21) that has a threaded leading end (22) and a stator (23) that has a central
cylindrical opening (25) and a frustroconical counterbore (24) that is adapted to receive the rotor. The protein solu-
tion/gas mixture is fed into this mill via opening (25). The gas and solution are mixed as they pass by the threads on the
shaft (22) and the mixture is emulsified and subjected to cavitation as it passes through the gap of the mill. The gap is
defined by the space between the conical surfaces of the rotor and stator.
[0046] Fig. 3 illustrates a Silverson mill. The structure of this mill is quite different from those of the mills of Figs 1 and
2. The depicted Silverson mill has a rotating shaft (30) that candies a paddle blade rotor (31). The rotor is received in a
cup-shaped perforated screen stator (32). The stator is mounted on a housing (33) fitted with an inlet fitting (34), The
inlet fitting (34) extends up into the housing (33) opening at the bottom center of the perforated screen stator (32). The
housing has a central opening (not shown) that communicates with the inlet fitting and with an opening (also not shown)
in the bottom of the stator. In this mill the solution/gas is fed via the inlet fitting into the bottom of the stator and is emul-
sified and cavitated between the flat faces (35) of the paddle rotor and the inner cylindrical surface of the stator. The
"gap" of this mill can be defined as the space between the rotor (31) and stator (32), but the effect of the gap size on
the process is influenced by the size of the perforations (36) of the stator.
[0047] After passing through the mill the product may be cooled, typically to 10 - 20°C, and defoamed by settling or
by adding biocompatible defoaming agents that do not adversely affect the microspheres.
[0048] Passing the mixture through such a mill or equivalent device emulsifies and cavitates the mixture to form micro-
spheres in the range of about 0.1 to 10 microns (mean diameter). Microsphere size may be determined by a suitable
particle counter, for example a Coulter Multisizer II (Coulter Electronics, Hialeah, Fl).
[0049] When using a mill such as those described in Figs. 1-3. the rotor speed, gap size and gas:liquid ratio are the
principal process parameters which affect the characteristics (mean size, size distribution, and concentration of micro-
spheres) of the microsphere product. Those parameters are adjusted empirically to provide a product having the
desired characteristics. For any given product, its characteristics are defined clinically. For instance, putative specifica-
tions for perfluoropropane microspheres used for myocardial perfusions are: mean size, 4 microns; size distribution.
90% under 10 microns; concentration. 7 x 10® to 2 x 10^ microspheres/mL.
[0050] The invention is further illustrated by the following examples. These examples are not intended to limit the
invention in any manner.
EXAMPLE 1
Mechanical Cavitation Process Temperature Monitorinqand Control for Human Serum Albumin
[0051 ] As described above, the protein solution is pre-heated before processing so that the process temperature can
reach and maintain the incipient denaturation temperature.
[0052] A typical method of practice is as follows:
[0053] A Model 2 1^" Bematek Colloid Mill (Fig. 2; Bematek Systems, Beverly MA), was piped so that the inlet port
was connected to a heat exchanger. Gas impermeable tubing was used to make the soft connections between the heat
exchanger hose barbs.
[0054] The outlet port from the process head was connected to a stainless steel post-process chiller.
[0055] Solution temperature was monitored at three sites (T1 , T2 and T3). The T1 thermocouple was mounted in a
EP0 633 030B1
Swagelok "Tee" between the pre-heat heat exchanger and the mill head to measure the feed temperature of the protein
solution. A second "Tee" for introducing gas was also placed at the feed port. The T2 thermocouple was placed inside
the exit from the process head, approximately 1 cm from the rotor and 2 cm from the shaft so that the temperature of
the process could be accurately measured. In this way, the two temperatures can be measured independently, the feed
temperature (T1) and the process temperature. (T2). and compared to determine the amount of heating of the solution
during processing.
[0056] For this example, U.S.P. albumin was diluted with normal saline to make up a 1% (w/v) solution. The denatur-
ation temperature was determined experimentally, as described, to be 7&*C. It was fed into the mill at 200 mL/min fol*
lowing degassing along with perfluoropropane at 100 mUmin (50% v/v). Differences between T1 and T2 of 10° to 15°C
were noted. In order to obtain a process temperature of 77**C (1**C below denaturation temperature), the feed temper-
ature was adjusted to a range of 62** to 67°C. Since the amount of heat generated will vary with different milling param-
eters, it is necessary to determine the difference between T1 and T2 with each change in milling parameters, (choice
of mill, mill settings, flow rate, gas:liquid ratio, etc.) In order to target the process temperature to avoid bulk denaturation
of the protein while successfully encapsulating the gas microbubbles with a thin shell of denatured protein.
The chiller-out temperature (T3) was also monitored, and for best results was targeted at 20''C.
EXAMPLE 2
Mechanical Cavitation Method of Makino Microspheres Containing Different Gases
[0057] Microspheres containing various gases were produced as follows: 5% human albumin solution (USP) was
deaerated under continuous vacuum for two hours. The vacuum was released by filling the evacuated vessel with the
gas of interest. Insoluble gases utilized include sulfur hexafiuoride, perfluoroethane, and perfluoropropane. Micro-
spheres containing more soluble gases, air. nitrogen, oxygen and argon, were also produced. The use of argon was
representative of a high molecular weight, but relatively soluble, gas. The albumin solution was adjusted to 68°C via an
in-line heat exchanger and pumped at 100 mL/min into a 2 1/2" (63.5 mm) colloid mill (Greerco. Hudson, NH, model
W250V or AF Gaulin, Everett, MA, model 2F). The specific gas, at room temperature, was added to the liquid feed just
upstream of the inlet port at a flow rate of 120-220 mL/min. The gap between the rotor and the stator was adjusted to
2/1 000th inch (0.005 cm) and the albumin solution was milled continuously at about 7000 rpm at a process temperature
of 73°C.
[0058] The dense white solution of microspheres thus formed was immediately chilled to a temperature of 10**C by a
heat exchanger, and collected in glass vials. The vials were immediately sealed. The material was characterized with
regard to concentration and size distribution using a Coulter Counter. The results are shown in Table 3 below.
Table 3
Concentration
(^spheres/mL)
Mean Size (microns)
Perfluoropropane
8.3x10^
3.8
Perfluoroethane
10.6x10®
4.0
Sulfur hexafiuoride
8.4x10®
3.9
Air
9.2x10^
3.4
Nitrogen
5.4 X 10^
5.0
Oxygen
6.1x10^
3.9
Argon
4.1 xlO^
3.5
EXAMPLE 3
Effect Qf Rotpr Speed ^nd Gap Size
[0059] A 1% albumin solution was combined (200 mL/min) with perfluoropropane (100 mL/min) at a 50% gas to liquid
(v/v) ratio. Microspheres were prepared according to the procedure described in Example 1 using varying rotor speeds
and gap sizes. The data obtained are shown in Table 4.
EP0 633 030B1
Table 4
Rotor TiD Soeed
(ft/min)
GaD fcm)
Concentration
(^spheres/mL)
Mean Size fmicrons)
3500
0.01
0.76x10**
13.4
4300
0.01
2.43x10^
9.6
4800
0.01
9.38x10^
3.8
9700
0.01
20.96x10®
4.3
5200
0.02
12.87x10®
5.0
7000
0.02
12.50x10®
3.4
8700
0.02
14.42x10®
3.0
9600
0.02
15.22x10®
2.9
[0060] These results show that concentration increases and mean size decreases with increasing rotor speed while
20 Increasing gap size decreases concentration.
EXAMPLE 4
Effect of Gas to Liquid RatiQ
25
[0061] A 0.5% Albumin solution (100 mL/min) was combined with perfluoropropane at 20. 50, 70 or 100 mL/min (20,
50. 70 or 100% gas to liquid vA^) using a Gaulin mill with an approximate gap of 0.012 and a rotor tip speed of 9950
ft/min (50.5 m/sec). The data obtained are shown in Table 5:
30
Table 5
Gas/Liquid % (v/v)
Gap (cm)
Concentration
(^spheres/mL)
Mean Size (microns)
20
0.03
6.54x10**
3.6
50
0.03
7.51 x 10®
4.3
70
0.03
8.76x10®
5.0
100
0.03
8.66x10®
5.9
[0062] These results show that both concentration and mean size increase with an increase in gas:liquid ratio.
EXAMPLE 5
45
Method of MaKipg Insoluble Gag-FiUed Microgpheres by Sonic Cavitation
[0063] Air. sulfur hexaf luoride and perfluoroethane microspheres were prepared by both batch and continuous ultra-
sound cavitation processes. A solution of human albumin, 5%, USP, was degassed under vacuum and stored under the
so specific gas. The continuous sonication process was performed as described by Cerny (USP 4,957,656), substituting
the insoluble gases for air. The batch process was performed utilizing a 3/4" (1 .9 mm) liquid processing horn (Sonics
and Materials. Danbury CT). Gas was passed through the horn and into the albumin such that during the entire process
air was excluded. The albumin was warmed to 73°C and sonicated for 5 sec at 20 KHz at 60 microns double amplitude,
using a Branson piezoelectric converter and power source (Branson Ultrasonics, Danbury CT). The product was imme-
55 dlately transferred to a glass vial and sealed under gas.
[0064] The product consisted of a thick, milky suspension of microspheres at concentrations of 1 .4 x 10® to 1 .0 x 10^
microspheres/mL with a mean size of 2.5 to 3.3 microns.
EP0 633030B1
EXAMPLE 6
Microscopic Examination of Microspheres
[0065] Albumin microspheres containing various gases were prepared as described in Example 2 or 5. Microscopic
examination of the products revealed a monodisperse suspension of spherical microspheres. The microspheres were
collapsed by application of high pressure in a syringe until the suspension clarified. In all cases, microscopic reexami-
nation revealed the presence of hyaline, membranous shells from the collapsed microspheres.
EXAMPLE 7
Pressure Resistance of Microspheres
[0066] Albumin microspheres containing various gases were prepared as described in Example 2. Ten mL of each
suspension was placed in a 10 mL glass gas-tight syringe (Hamilton, Reno NV) fitted with a pressure gauge. All head-
space was removed and the apparatus was sealed. A constant pressure of 40 psig (2.76 bar) was applied for 3 min. A
Coulter Counter was then used to measure the sample particle concentration and distribution. Comparisons of the data
(Figures 4a-4e) before and after pressurization demonstrated a relative resistance of the insoluble gas microspheres to
40 psig (2.76 bar).
EXAMPLE 8
Pressure Resistance of Dilute Suspensio ns of Microspheres
[0067] Microspheres containing various gases were prepared as described in Example 2. Each sample of micro-
spheres was diluted to an equal volume of encapsulated gas per mL of phosphate-buffered saline (0. 1 5M), about a 1 :60
dilution. The diluted suspension was subjected to instant static pressures of 0.5 psig (0.035 bar) to 7.5 psig (0.52 bar)
in a sealed vessel with adequate head space. Figure 5 shows the effect of pressure on microsphere concentration.
Microspheres containing the insoluble gases perfluoropropane, perf luoroethane and sulfur hexafluoride are much more
pressure-resistant than air or high molecular weight argon-filled microspheres of the same concentration and size dis-
tributions (Figure 6). Physiological pressures in the bloodstream range from a peripheral venous pressure of 1.5 psig
(0,10 bar) to 2.5 psig (0.17 bar) in the myocardial wall.
EXAMPLE 9
Effect of Degassed Buffer on Microspheres
[0068] Albumin microspheres containing various gases were prepared as described in Example 2. Phosphate-buff-
ered saline (PBS) was degassed by boiling just before use. 0.05 mL to 1 .5 mL aliquots of the hot buffer were placed in
13 x 100 test tubes and allowed to cool 1 min to room temperature in a water bath, A constant volume of microspheres
was added to each tube. After mixing, the final volume was brought to 3.0 mL with PBS and the microsphere concen-
tration was determined. Figure 7 shows that improved survival in degassed solutions is obtained for the microspheres
containing the insoluble gases perfluoropropane, perfluoroethane and sulfur hexafluoride.
[0069] Microspheres containing air, sulfur hexafluoride or perfluoroethane were diluted into whole blood. Air-filled
microspheres exhibited collapse. The insoluble gas-filled microspheres were shown to survive dilution in fresh whole
blood.
EXAMPLE 10
El a^t i Q ' ty
[0070] Microspheres prepared from various gases were prepared as described in Example 2. Microspheres were
diluted into phosphate-buffered saline, as described in Example 8, and placed in a clear cell positioned on the stage of
a microscope. The cell was connected to a nitrogen source that allowed for observing the effects of rapid application
and release of physiological pressures on the microspheres.
[0071 ] Tlie application of 1 .5 psig (0. 1 0 bar) or greater to the soluble gas containing microspheres resulted in observ-
ing the complete loss of spherical bodies. The microspheres did not reform upon the release of the pressure, indicating
irreversible destruction. The application of less than 1 .5 psig (0. 1 0 bar) resulted in deformation and wrinkling of the shell
EP0 633 030B1
with incomplete loss of microspheres. The spherical appearance or population could not be restored upon release of
the applied pressure.
[0072] The application of pressure up to several psig to a suspension of microspheres containing the insoluble per-
fluorocarbon gases resulted in a reduction of the diameter of the microspheres. The diameter of the microspheres
5 returned to the original dimensions upon the release of the pressure.
[0073] Sulfur hexafluoride microspheres also exhibited enhanced elasticity under applied physiological pressure rel-
ative to air-filled microspheres, but less elasticity relative to the perfluorocarbon microspheres.
[0074] These observations indicate that the microspheres containing insoluble gases were not only resistant to pres-
sure, but recovered after pressure was released. This is indicative of an elastic protein shell.
10
PROCESS METHODS
A) Manual Sonication: Open System (Equivalent to EPA 554,213 One-Step Method)
15 [0075] The method described in U.S. Patent No. 4,844,882 and European Patent Application 554,213 was used to
prepare microspheres as follows:
[0076] A 20 cc syringe barrel was fitted with a T-type thermocouple inserted through the tip and mounted onto a sup-
port stand. The syringe was filled to the 16 cc mark with Swiss Red Cross 5% human serum albumin. Gas (perfluoro-
propane (C3F8) or sulfur hexafluoride (SFe)) was introduced into the top of the syringe barrel and flowed over the
20 surface of the liquid. A sonicating horn was lowered to the 10 cc mark, below the surface of the solution, and operated
at 50% power until the temperature of the solution rose to 72.8 - 73''C; approximately 1 minute. The horn was immedi-
ately withdrawn to the meniscus ± 1 mm and the power level increased to 65%. Sonication continued for 5 seconds,
with an additional temperature increase of 1 .2 - 2''C. The product was poured into a glass vial to capacity and sealed.
25 B) Continuous Sonication: Closed System
[0077] The method described in U.S. Patent No. 4,957,656 was used to prepare perf luoropropane and sulfur hexaflu-
oride microspheres as follows:
[0078] Human serum albumin was diluted to a 1% w/v solution with sterile saline. The solution was heated to incipient
30 denaturation, approximately 76°C. The system was closed to the external atmosphere and perfluoropropane or sulfur
hexafluoride gas was introduced into the liquid flow (1 :1} in place of air. The product was made continuously by flowing
the gas/albumin mixture past the sonicator horn at approximately 100 ml liquid/min. The product was chilled upon exit
from the sonication chamber by passage through a heat exchanger and collected as a bulk liquid suspension of micro-
spheres. Handling and storage conditions were similar to that given for manually produced microspheres.
35
C) Mechanical Cavitation: Closed System
[0079] Albumin microspheres containing perfluoropropane or sulfur hexafluoride gas were also produced in a closed
system by milling a mixture of 1% human serum albumin and gas, similar to that described in Example 2. Albumin sotu-
40 tlon, heated to a temperature sufficient to allow microsphere formation by the mechanical cavitation of a given mill, was
mixed 1 :1 (v:v) gas and introduced into a colloid mill. The liquid flow rate was dependent upon the capacity or size of
the mill, typically 100 to 500 ml/min. A Silverson L4R mill and a Bematek 3" (76.2 mm) production colloid mill were used
for this evaluation. The outflow from the mill was cooled by passage through a heat exchange system and the resulting
albumin microsphere suspension was collected in bulk. The product was filled into glass vials, similar to the other proc-
45 esses.
ANALYTICAL METHODS
A) Population Dynamics
50
[0080] Population dynamics were evaluated with a Coulter Multisizer II using a 50 micron aperture. Albumin micro-
spheres prepared as described in PROCESS METHODS were diluted 1 :10,000 into Isoton and a 500 ^ sample was
analyzed. Concentration, mean size, and encapsulated gas volume per ml of the original microsphere suspension were
obtained.
55
B) Gas Content
[0081]
The percentage of perfluoropropane entrapped in duplicate lots of microspheres prepared as described in
EP0 633 030B1
PROCESS METHODS was determined by gas chromatography on a Hewlett Packard 5890. A sample of the micro-
sphere suspension was taken in a gas tight syringe. The gas was released from the microspheres using an anti-foam
agent in ethanol and the entrapped gas was detected by thermal conductivity.
5 C) Pressure Resistance
[0082] Pressure resistance of albumin microspheres was evaluated by a method similar to that reported by Sintetica
in European Patent Application 554,213. Microspheres were diluted in aerated phosphate buffered saline, to approxi-
mately 1 absorbance unit at 600 nm, in a 3 ml pressure cuvette. The neck was attached to a pressure source and the
10 cuvette placed in a recording spectrophotometer. The pressure in the cuvette was increased linearly from 0 to 5 or 10
psig (0.35 to 0.69 bar) over 150 seconds, at which time the pressure was released. The pressure ramp was created by
a proportioning solenoid valve (Honeywell) and a pressure transducer (Omega) that were placed between a 20 psi
(1 .38 bar) pressure source (Ng tank) and a 5 liter stainless steel reservoir. The cuvette was connected to the steel res-
ervoir through a digital pressure gauge. A PC-type computer equipped with an analog to digital converter and a digital
15 to analog converter board (National Instruments) controlled the opening of the valve and read the pressure transducer:
The reservoir, and cuvette, was pressurized at a selected rate until the desired pressure was achieved. The optical den-
sity of the microsphere suspension was monitored as a function of time and pressure. The data was corrected for the
natural flotation rate of microspheres in the cuvette.
20 RESULTS
A) Population Dynamics
[0083] Albumin microspheres produced by the methods of manual sonication. continuous sonlcatlon and mechanical
25 cavitation were analyzed for concentration, mean size, encapsulated gas volume and size distribution within 24 hours
after manufacture. All measurements were performed in duplicate, as a minimum, and are presented as the average.
The results of these measurements are given in Table 6.
Table 6
35
Gas
Method
Cone. (1tf*/ml)
Mean Size dim)
Vol (ml/ml)
SFe
Manual Sonication
8.7
3.2
0.046
SFe
Continuous Sonication
12.7
2.7
0.034
SFe
Mechanical Cavitation
10.0
3.8
0.054
C3F8
Manual Sonication
13.4
2.8
0.033
Manual Sonication
17.7
2.8
0.056
C3F8
Continuous Sonication
10.1
3.0
0.050
C3F8
Continuous Sonication
6.7
4.3
0.127
C3F8
Mechanical Cavitation (Bematek Mill)
31.0
3.0
0.23
C3F8
Mechanical Cavitation (Silverson Mill)
6.9
5.0
0.34
Microspheres produced by all methods were stable for the duration of this study, at least several weeks at 4°C.
B) Gas Content
50
[0084] Analyses of the composition of entrapped perfluoropropane gas in duplicate lots of microspheres are given in
Table 7.
55
EP0 633 030B1
Table 7
Method*
%C3F8
Manual Sonication
70.0
Continuous Sonication
89.5
Mechanical Cavitation
95.5
* Average results of duplicate lots.
[0085] These results demonstrate that microspheres made in the open system using manual sonication encapsulate
much less of the gas used to form the microspheres than those made in the closed systems (continuous sonication and
mechanical cavitation.) The microspheres made in the closed system were made in the absence of oxygen, as deter-
mined using an oxygen electrode. Microspheres made by all three methods were subjected to the same amount of
exposure to the atmosphere during handling and sampling (which accounts for less than 100% perfluoropropane gas
being measured in microspheres made using the two closed system procedures), thus there was oxygen (and other
atmospheric gases) present during formation in the open system which diminished the efficiency of gas encapsulation.
C) Pressure Resistance
[0086] A suspension of gas-filled microspheres will decrease in optical density with increasing pressure due to a
decrease in size and associated change in surface area. Shrinkage is due to two factors; reversible cortipression
according to the gas laws, and in-eversible loss of the gas core to the surrounding liquid due to increased solubility
according to Henry's law. Upon the release of an applied pressure, only that fraction of the volume loss due to compres-
sion is recovered, and which can be observed by an increase in optical density. The loss of entrapped gas to the sur-
rounding solution does not reenter the microspheres upon depressurization, but is lost to the head space above the
solution.
[0087] Fig. 8 shows the result of imposing a linear pressure gradient up to 10 psi (0.69 bar) on 1 OD suspensions of
albumin microspheres prepared with perfluoropropane gas by the manual sonication (open system) method as well as
the continuous sonication and mechanical cavitation (closed system) methods. Both closed system methods yielded
microspheres that exhibited compression with increasing pressure, with a total recovery of volume upon release of the
pressure at the end of the gradient. Loss of entrapped gas to the surrounding solution was not observed. Albumin
microspheres prepared in the open system (manual sonication method) exhibited greater compression with applied
pressure and only a partial recovery of volume upon release of pressure due to the irreversible loss of the gas core,
resulting in a 40% destruction of microspheres.
Claims
1 . A method of making encapsulated gas microspheres useful as an ultrasonic imaging agent, the method comprising
subjecting a mixture of an aqueous solution of a f ilmogenic protein and a pharmacologically acceptable gas to ultra-
sonic or mechanical cavitation characterised in that the method is conducted in the absence of oxygen and the gas
has a solubility, in water at 25''C, of less than 0.01 mL of gas per mL of water.
2. A method according to claim 1 wherein the protein is human serum albumin and the gas is perfluoropropane.
3. A method according to claim 1 or 2 wherein the pharmacologically acceptable gas has a diffusivity in water at 25**C
of less than 4x10"^ cm^/sec.
4. A method according to claim 1 or 2 wherein the pharmacologically acceptable gas is perf luorobutane.
5. A method according to claim 1 or 2 wherein the pharmacologically acceptable gas is sulfur hexaf luoride.
6. A method according to claim 1 or 2 wherein the pharmacologically acceptable gas is perfluoroethane.
7. A method according to claim 1 or 2 wherein the pharmacologically acceptable gas is perfluoromethane.
EP0 633 030B1
PatentansprQche
1. Verfahren zur Herstellung von eingekapselten, Gas enthaltenden Mikrokugelchen, die als Ultraschallabbildungs-
mittel verwendbar sind, wobei mit einem Gemisch einer wdf3rigen LOsung eines f ilmbildenden Proteins und eines
pharmakologisch vertrdglichen Gases eine Hohlraumbiidung mittels Uttraschall Oder eine mechanische Holilraum-
bildung durchgefuhrt wird, dadurcli gekennzeichnet, daf3 das Verlaiiren in Abwesenheit von Sauerstoff durchge-
fuhrt wird und das Gas in Wasser bei 25°C eine LOsliclikeit von weniger als 0,01 ml Gas/ml Wasser besitzt.
2. Verfahren nach Anspruch 1 , wobei das Protein menschliches Serumalbumin und das Gas Pert luorpropan ist.
3. Verfahren nach Anspruch 1 oder 2, wobei das pharmakologisch vertragliche Gas eine Diffusion von weniger als 4
X 10"^ cm^/sec in Wasser bei 25**C besitzt.
4. Verfahren nach Anspruch 1 oder 2, wobei das pharmakologisch vertragliche Gas Perfluorbutan ist.
5. Verfahren nach Anspruch 1 oder 2, wobei das pharmakologisch vertragliche Gas Schwefelhexafluorid ist.
6. Verfahren nach Anspruch 1 oder 2, wobei das pharmakologisch vertragliche Gas Pert luorethan ist.
7. Verfahren nach Anspruch 1 oder 2, wobei das pharmakologisch vertragliche Gas Perfluormethan ist.
Revendications
1 . Proc6d6 de pr6paration de microspheres de gaz encapsul6, utiles en tant qu'agent d'imagerie ultrasonique, ce pro-
c^6 ayant pour objet de soumettre un melange d'une solution aqueuse d'une prot^ine f ilmog^ne et d'un gaz phar*
macologiquement acceptable a une cavitation ultrasonique ou m^canique. ce proc6d6 6tant caract^ris^ en ce qu1l
est mis en oeuvre en I'absence d'oxyg^ne et en ce que le gaz a une solubility, dans de I'eau k 25 ""C, inf^rieure a
0,01 ml de gaz par ml d'eau.
2. Proc^e selon la revendication 1 , dans lequel la prot^ine est la s6rumalbumine humaine et le gaz est le perf luoro-
propane.
3. Proc^y selon la revendication 1 ou 2, dans lequel le gaz pharmacologiquement acceptable a une diffusivitd dans
I'eau a 25 °C inf6rieure a 4 x 10'^ cm^/s.
4. Proc^6 selon la revendication 1 ou 2. dans lequel le gaz pharmacologiquement acceptable est le perf luorobutane.
5. Proc§d6 selon la revendication 1 ou 2. dans lequel le gaz pharmacologiquement acceptable est I'hexaf luorure de
soufre.
6. Proc6d6 selon la revendication 1 ou 2, dans lequel le gaz pharmacologiquement acceptable est le pert luoroathane.
7. Proc6d6 selon la revendication 1 ou 2, dans lequel le gaz pharmacologiquement acceptable est le perfluorom6-
thane.
EP0 633 030B1
EP0 633 030B1
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EP0 633 030 B1
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EPO 633 030 B1
Fig. 5
8.9
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Fig. 6
EP0 633 030B1
Degassed Buffer (mL)
Fig. 7
(mi 009) AXisNaa tydimo
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
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Understanding 9/11